Project Summary (Abstract) Approximately 170 million people are chronically infected with hepatitis C virus (HCV). The virus causes about a million deaths and 2-3 million new infections each year. HCV establishes chronic hepatotropic infection in the majority of infected individuals. However, not all chronically infected persons develop liver diseases. The viral and host factors, and their interactions, that determines the development of HCV persistence and liver diseases remains obscure largely due to the lack of an appropriate small animal model. Notably, HCV induced liver diseases develops after years or decades of persistent viral infection in humans, the natural host of HCV, and therefore an informative liver disease model should develop long term viral persistence leading to chronic liver inflammation and diseases. Only chimpanzees can serve as immunocompetent hosts to study HCV infection but are unavailable. Considering the strict species tropism of HCV, the best alternative is to develop a surrogate model where the virus and its infection in the host mirror HCV infection in humans and chimpanzees. GBV-B served as an informative surrogate model but it mostly causes acute resolving infection (of a few months). Rodent hepaciviruses (RHV) are animal homologs of HCV. We isolated a RHV from wild rats (Rattus norvegicus), designated RHV-rn1 (preliminary data). We determined that RHV-rn1 possesses HCV?s defining hallmarks: hepatotropism, propensity to life-long persistence and slow progressing liver disease. Immune responses elicited by RHV-rn1 and HCV are similar including the induction of interferon stimulated genes in the liver during acute and chronic viral infection Taken together, we showed that RHV-rn1 infection of immunocompetent rats is an innovative model to study HCV-related persistence and pathogenesis. We propose to characterize the host factors that dictate viral infection outcomes and diseases. Since, their better characterization is critical for developing new means of preventing viral persistence and diseases. Aim-1 is to study natural history of virus pathogenesis and dynamics of innate immune responses culminating in different infection outcomes and liver pathologies. Aim-2 is high-resolution mapping of innate immune responses in the liver of infected rats. We will study serial gene expression changes using cell-type specific and single-cell RNA-seq analysis of virus infected and uninfected hepatocytes, sinusoidal endothelial cells, Kupffer cells and natural killer cells. Aim-3 is to target the viral genomes and specific innate immune responses for achieving virus clearance or averting liver diseases. We will use miRNA expressing Adeno-associated virus (AAV) vectors to inhibit virus replication or repress/de- repress host gene expression. Consequently, we will know the precise role of the targeted host responses in hepacivirus persistence and pathogenesis. We believe that the proposed studies will provide unprecedented insights into the in vivo relevance of host responses during HCV pathogenesis. Additionally, further characterization of our novel fully immunocompetent and tractable model will allow development of innovative means to prevent viral persistence and liver diseases. !